Polyclonal-monoclonal ELISA assay for detecting N-terminus pro-BNP

ABSTRACT

A specific and sensitive in vitro ELISA assay and diagnostic test kit is disclosed for determining levels of NT-proBNP protein in a variety of bodily fluids, non-limiting examples of which are blood, serum, plasma, urine and the like. The NT-proBNP ELISA assay test employs the sandwich ELISA technique to measure circulating NT-proBNP in human plasma. In order to obtain antibodies with specific binding properties for targeted amino acid sequences within human proBNP, recombinant human proBNP (or rhproBNP) was expressed and purified for use as an immunogen. Polyclonal antibodies (PAb) to specific amino acid sequences were subsequently purified from goat serum by sequential affinity purification. Monoclonal antibodies were raised against specific polypeptides. Recombinant human NT-proBNP (or rhNT-proBNP) was expressed and purified in order to obtain material for use in calibration of a quantitative method for measurement of human NT-proBNP.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/236,447, filed on Sep. 19, 2011, now issued as U.S. Pat. No.9,371,382, which is a continuation of U.S. patent application Ser. No.12/421,409, filed on Apr. 9, 2009, now abandoned, which is acontinuation of U.S. patent application Ser. No. 11/375,432, filed onMar. 13, 2006, now issued as U.S. Pat. No. 7,527,939, which is acontinuation of U.S. patent application Ser. No. 10/359,051, filed onFeb. 4, 2003, now abandoned, which is a continuation of U.S. patentapplication Ser. No. 10/300,733, filed on Nov. 18, 2002, now abandoned,the contents of these related applications are herein incorporated byreference.

FIELD OF THE INVENTION

This invention relates to an NT-proBNP protein ELISA assay procedure andtest kit which is a specific and sensitive in vitro assay for measuringthe concentration of NT-proBNP in bodily fluids, particularly humanplasma. The invention particularly relates to an NT-proBNP protein ELISAassay having a particularly high diagnostic specificity, whereby theassay is particularly designed to be predictive of mortality as a resultof congestive heart failure.

BACKGROUND OF THE INVENTION

B-type natriuretic peptide (Brain natriuretic peptide, BNP) belongs tothe family of structurally similar, but genetically distinct natriureticpeptides (NPs) first described by de Bold et al. (de Bold A J. Heartatria granularity: effects of changes in water-electrolyte balance. ProcSoc Exp Biol Med 1979; 161:508-511; de Bold A J, Borenstein H B, VeressA T and Sonnenberg H. A rapid and potent natriuretic response tointravenous injection of atrial myocardial extracts in rats. Life Sci1981; 28:89-94).

The NPs possess potent diuretic, natriuretic and vasodilatory propertiesand have been reported as valuable diagnostic and prognostic markers incardiovascular disease, particularly for patients in New York HeartAssociation (NYHA) classes I-IV congestive heart failure (CHF) (BoomsmaF and van den Meiracker A H. Plasma A- and B-type natriuretic peptides:physiology, methodology and clinical use. Cardiovasc Res 2001;51:442-449).

The BNP gene encodes for a 108 amino acid residue precursor molecule,proBNP (Sequence ID No. 1). Prior to secretion by cardiomyocytes,cleavage of this prohormone results in the generation of bioactive BNPfrom the COOH terminus. In 1995, Hunt et al. (Hunt P J, Yandle T G,Nicholls M G, Richards A M and Espiner E A. The Aminoterminal Portion OfProbrain Natriuretic Peptide (Probnp) Circulates In Human Plasma.Biochem Biophys Res Commun 1995; 14:1175-1183; Hunt P J, Richards A M,Nicholls M G, Yandle T G, Doughty R N and Espiner E A. ImmunoreactiveAmino-Terminal Pro-Brain Natriuretic Peptide (NT-PROBNP): A New MarkerOf Cardiac Impairment. Clin Endocrinol 1997; 47:287-296) demonstratedthat fragments corresponding to the N-terminal portion of the cleavedprohormone, NT-proBNP, also circulated in plasma, and like BNP, were apotentially important, and possibly more discerning, marker ofventricular dysfunction. Many studies have demonstrated the clinicalutility of measuring plasma concentrations of NPs, including NT-proBNP.NPs have been suggested as the biomarkers of choice for diagnosis andrisk stratification of patients with heart failure (Clerico A, Del Ry Sand Giannessi D. Measurement Of Cardiac Natriuretic Hormones (AtrialNatriuretic Peptide, Brain Natriuretic Peptide, And Related Peptides) InClinical Practice: The Need For A New Generation Of Immunoassay Methods.Clin Chem 2000; 46:1529-1534: Mair J, Hammerer-Leacher A and PuschendorfB. The Impact Of Cardiac Natriuretic Peptide Determination On TheDiagnosis And Management Of Heart Failure. Clin Chem Lab Med 2001;39:571-588; Sagnella G A. Measurement And Importance Of Plasma BrianNatriuretic Peptide And Related Peptides. Ann Clin Biochem 2001;38:83-93; Selvais P L, Donckier J E, Robert A, Laloux O, van Linden F,Ahn S, Ketelslegers J M and Rousseau M F. Cardiac Natriuretic PeptidesFor Diagnosis And Risk Stratification In Heart Failure: Influences OfLeft Ventricular Dysfunction And Coronary Artery Disease On CardiacHormonal Activation. Eur J Clin Invest 1998; 28:636-642; McDonagh T A,Cunningham A D, Morrison C E, McMurray J J, Ford I, Morton J J andDargie H J. Left Ventricular Dysfunction, Natriuretic Peptides, AndMortality In Urban Population. Heart 2001; 86:21-26). Several studieshave shown the utility of using NP measurements to identify patientswith left ventricular dysfunction, even amongst patients who areasymptomatic (i.e. NYHA class I) and it has been suggested that NPmeasurements as a screening tool may help effectively target patientswithin high risk heart failure groups (e.g. coronary artery disease,hypertension, diabetes, aged) who will require follow-up assessment andtreatment (Hughes D, Talwar S, Squire I B, Davies J E and Ng L L. AnImmunoluminometric Assay For N-Terminal Pro-Brain Natriuretic Peptide:Development Of A Test For Left Ventricular Dysfunction. Clin Sci 1999;96:373-80; Omland T, Aakvaag A, Vik-Mo H. Plasma Cardiac NatriureticPeptide Determination As A Screening Test For The Detection Of PatientsWith Mild Left Ventricular Impairment. Heart 1996; 76:232-237; McDonaghT A, Robb S D, Murdoch D R, Morton J J, Ford I, Morrison C E, et al.Biochemical Detection Of Left-Ventricular Systolic Dysfunction. Lancet1998; 351:9-13; Schulz H, Langvik T A, Lund Sagen E, Smith J, Ahmadi Nand Hall C. Radioimmunoassay For N-Terminal Probrain Natriuretic PeptideIn Human Plasma. Scand J Clin Lab Invest 2001; 61:33-42; Talwar S,Squire I B, Davies J E, Barnett D B and Ng L L. Plasma N-TerminalPro-Brain Natriuretic Peptide And The ECG In The Assessment OfLeft-Ventricular Systolic Dysfunction In A High Risk Population. EurHeart J 1999; 20:1736-1744; Hystad M E, Geiran O R, Attramadal H,Spurkland A, Vege A, Simonsen S and Hall C. Regional Cardiac ExpressionAnd Concentration Of Natriuretic Peptides In Patients With SevereChronic Heart Failure. Acta Physiol Scand 2001; 171:395-403; Hobbs F DR, Davis R C, Roalfe A K, Hare R, Davies M K and Kenkre J E. ReliabilityOf N-Terminal Pro-Brain Natriuretic Peptide Assay In Diagnosis Of HeartFailure: Cohort Study In Representative And High Risk CommunityPopulations. BMJ 2002; 324:1498). NPs have been shown to have goodprognostic value with regards to both morbidity and mortality in heartfailure. Several studies have also demonstrated the utility of NPmeasurements in the prediction of left ventricular dysfunction andsurvival following acute myocardial infarction (Richards A M, Nicholls MG, Yandle T G, Frampton C, Espiner E A, Turner J G, et al. PlasmaN-Terminal Pro-Brain Natriuretic Peptide And Adrenomedullin. NewNeurohormonal Predictors Of Left Ventricular Function And PrognosisAfter Myocardial Infarction. Circulation 1998; 97:1921-1929; Luchner A,Hengstenberg C, Lowel H, Trawinski J, Baumann M, Riegger G A J, et al.N-Terminal Pro-Brain Natriuretic Peptide After Myocardial Infarction. AMarker Of Cardio-Renal Function. Hypertension 2002; 39:99-104; CampbellD J, Munir V, Hennessy O F and Dent A W. Plasma Amino-Terminal Pro-BrainNatriuretic Peptide Levels In Subjects Presenting To The EmergencyDepartment With Suspected Acute Coronary Syndrome: Possible Role InSelecting Patients For Follow Up? Intern Med J 2001; 31:211-219; NilssonJ C, Groenning B A, Nielsen G, Fritz-Hansen T, Trawinski J, HildebrandtP R, et al. Left Ventricular Remodeling In The First Year After AcuteMyocardial Infarction And The Predictive Value Of N-Terminal Pro BrainNatriuretic Peptide. Am Heart J 2002; 143:696-702). Monitoring NP levelsmay also provide guidance in tailoring therapies to meet the requiredintensity of the individual patient and in monitoring therapeuticefficacy (Richards A M, Doughty R, Nicholls G, MacMahon S, Sharpe N,Murphy J, et al. Plasma N-Terminal Pro-Brain Natriuretic Peptide AndAdrenomedullin. Prognostic Utility And Prediction Of Benefit FromCarvedilol In Chronic Ischemic Left Ventricular Dysfunction. J Am CollCardiol 2001; 37:1781-1787; Troughton R W, Frampton C M, Yandle T G,Espiner E A, Nicholls M G and Richards A M. Treatment Of Heart FailureGuided By Plasma Aminoterminal Brain Natriuretic Peptide (N-BNP)Concentrations. Lancet 2000; 355:1126-30).

PRIOR ART

WO 93/24531 (U.S. Pat. No. 5,786,163) to Hall describes an immunologicalmethod of identifying N-terminal proBNP and the antibodies used for it.To obtain these antibodies single synthetically produced peptides fromthe sequence of N-terminal proBNP are used. The production of antibodiesby means of peptide immunization is possible in principle but theaffinity regarding the whole molecule generally is too low to reach thenecessary sensitivity in a test procedure. In addition, there is adanger that when using peptides the antibodies obtained can, forexample, identify the C-terminus of the peptide and can therefore onlybind to this fragment of the whole molecule, thus resulting inantibodies which generally cannot bind to the whole molecule, or can doso to only a limited extent. In WO 93/24531 an antibody against onesingle peptide derived from the N-terminal proBNP is produced. It isshown that the antibodies produced bind to the immunization peptide(amino acids 47-64) in the competitive test format. It is however notshown that the antibodies are able to bind to native N-terminal proBNPas a whole molecule in a sample. Additionally, the sandwich testdescribed in WO 93/24531 in a sample cannot be performed as describedsince there was no appropriate standard material and no antibodiesagainst two different epitopes. Additionally, the competitive testperformed in PCT 93/24531, where the peptide 47-64 competes in alabelled form as a tracer with a sample or the unlabelled peptidestandard 47-64 to bind to polyclonal antibodies from rabbit serum,suffers from the fact that only a very moderate competition is reachedafter 48 hours of incubation from which only a low detection limit ofapprox. 250 fmol/ml can be derived. This is neither sufficient for thedifferentiation of healthy individuals and patients suffering from heartfailure nor for a differentiated classification of patient samples intothe severity degrees of heart failure. In addition, the long incubationtimes of the competitive test are not acceptable for routinemeasurements of the samples in automated laboratories.

Hunt et al. (Clinical Endocrinology 47 (1997), 287-296) also describes acompetitive test for the detection of N-terminal proBNP. For this acomplex extraction of the plasma sample is necessary before themeasurement; this may lead to the destruction of the analyte and errormeasurements. The antiserum used is produced analogously to WO 93/24531by immunization with a synthetic peptide—Hunt et al. produces theantiserum by immunization with the N-terminal proBNP amino acids 1-13and the peptide of amino acids 1-21 is used as a standard. For this testlong incubation times are necessary too. After an incubation of 24 hoursa lower detection limit of 1.3 fmol/ml is reached.

WO 00/45176, Method of Identifying N-Terminal proBNP, Karl et al.,discloses monoclonal and polyclonal antibodies isolated via the use of arecombinant NT-proBNP immunogen. The reference suggests the formation ofan assay using the disclosed antibodies as being specific for NT-proBNPin bodily fluids. As will be more fully described, a comparison of thearea under the curve (AUC) of a plot of the Receiver OperatedCharacteristics (ROC) for this assay versus the assay of the instantinvention indicates that the instant invention demonstrates superiordiagnostic performance.

WO 00/35951, Natriuretic Peptide Fragments, is directed toward an assayfor NT-proBNP utilizing two antibodies directed toward differingepitopes of the NT-proBNP sequence. This assay suffers from similardeficiencies as that of Hall (U.S. Pat. No. 5,786,163) in that theantibodies are raised against synthetic peptide fragments as theimmunogen.

SUMMARY OF THE INVENTION

The instantly disclosed NT-proBNP protein ELISA assay and test kit is aspecific and sensitive in vitro assay that is capable of measuring theconcentration of NT-proBNP in a variety of bodily fluids, non-limitingexamples of which are blood, serum, plasma, urine and the like. Thefollowing examples and descriptions will exemplify the use of the assayin human plasma.

As used herein, the term “antibody or antibodies” includes polyclonaland monoclonal antibodies of any isotype (IgA, IgG, IgE, IgD, IgM), oran antigen-binding portion thereof, including but not limited to F(ab)and Fv fragments, single chain antibodies, chimeric antibodies,humanized antibodies, and a Fab expression library.

The NT-proBNP ELISA assay test employs the sandwich ELISA technique tomeasure circulating NT-proBNP in human plasma.

In order to obtain antibodies with specific binding properties fortargeted amino acid sequences within human proBNP, recombinant humanproBNP (or rhproBNP) was expressed and purified for use as an immunogen.Polyclonal antibodies (PAb) specific for amino acid sequences withinproBNP (1-25, 26-51, 52-76 or 77-108) of Sequence ID No. 1 weresubsequently purified from goat serum by sequential affinitypurification In order to obtain material for use in calibration of aquantitative method for measurement of human NT-proBNP, recombinanthuman NT-proBNP (or rhNT-proBNP) was expressed and purified. Monoclonalswere produced from supernatants for use in an NT-proBNP ELISA in pairingwith the instantly described Goat Polyclonal Antibodies. The monoclonalswere biotinylated and used as a detector antibody to bind to theNT-proBNP protein bound to anti-NT-proBNP capture antibody, thus forminga sandwich.

Accordingly, it is an objective of the instant invention to provide goatpolyclonal antibodies raised against recombinant human proBNP, whichantibodies are specifically selected to exhibit a specific affinity fortargeted amino acid sequences within human proBNP.

It is a further objective of the instant invention to provide aquantitative method for measurement of human NT-proBNP, whereby adiagnostic/screening tool for accurately predicting mortality incongestive heart failure patients may be determined.

It is yet an additional objective of the instant invention to providemonoclonal antibodies useful in providing a particularly sensitive andspecific in vitro diagnostic assay when combined with the previouslyselected goat polyclonal antibodies.

It is still an additional objective of the instant invention to providean ELISA Test Kit for the purpose of carrying out the above-outlineddiagnostic/screening procedure to determine levels of NT-proBNP.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the method of selection of NT-proBNP and targetpeptides starting from a pre-proBNP precursor protein;

FIG. 2 is an ROC curve for the goat polyclonal/6G11 monoclonal assay;

FIG. 3 is a box-plot of NT-proBNP levels in NYHA Class III and IV versuscontrols;

FIG. 4 is a box-plot of NT-proBNP levels in control subjects, stratifiedby age;

FIG. 5 outlines the ELISA procedure for utilizing the goatpolyclonal/6G11 monoclonal assay of the instant invention.

DETAILED DESCRIPTION OF THE INVENTION

The NT-proBNP ELISA assay test employs the sandwich ELISA technique tomeasure circulating NT-proBNP in human plasma. Microplate wells coatedwith goat polyclonal anti-NT-proBNP capture protein constitute the solidphase. Test subject plasma, standards and controls are added to thecoated wells and incubated with incubation buffer. No sample extractionstep is required. If NT-proBNP protein is present in the test sample, itwill be captured by NT-proBNP specific antibody coated on the wells.After incubation and washing, a monoclonal anti-NT-proBNP detectorantibody is added to the wells. The detector antibody binds to theNT-proBNP protein, or immunogenic fragments thereof, e.g. polypeptidefragments which are recognized by said antibody, bound to anti-NT-proBNPcapture antibody, thus forming a sandwich. After incubation and washing,a polyclonal donkey anti-mouse IgG labeled with horseradish peroxidase(HRP) is added to the wells. Following incubation and washing, an enzymesubstrate is added to the wells and incubated. An acidic solution isthen added in order to stop the enzymatic reaction. The degree ofenzymatic activity of immobilized HRP is determined by measuring theoptical density of the oxidized enzymatic product in the wells at 450nm. The absorbance at 450 nm is proportional to the amount of NT-proBNPin the test subject sample. A set of NT-proBNP protein standards is usedto generate a standard curve of absorbance versus NT-proBNPconcentration from which the NT-proBNP concentrations in test specimensand controls can be calculated. It is understood that detection of theimmunoreaction may be accomplished via direct or indirect methods whichare well-known in the art.

In order to obtain antibodies with specific binding properties fortargeted amino acid sequences within human proBNP, recombinant humanproBNP (or rhproBNP) was expressed and purified for use as an immunogen.ProBNP-pUC9 plasmid construct was obtained from Dr. Adolfo J. de Bold(Ottawa Heart Institute). The full-length rhproBNP open reading frame(ORF) was obtained by polymerase chain reaction (PCR) and subcloninginto pET32c (NcoI/XhoI). The pET32c vector was modified by removing 81nucleotides so that the final fusion protein would not contain the S-tagand enterokinase sites. The sequence at the N-terminus of the rhproBNPORF consisted of thioredoxin and poly-histidine tags and a thrombincleavage site. There was no extra sequence at the C-terminus. Theprotein was expressed in Escherichia coli BL21 (DE3) cells and the crudecellular extract was prepared in non-denaturing conditions. Thesubsequent affinity purification was completed by Ni-NTA chromatographyfollowing the supplier's recommendations. Prior to injections, endotoxinlevels in the rhproBNP solutions were lowered to acceptable levels usinga Detoxigel® endotoxin-removing resin following the supplier'srecommendations.

Polyclonal Antibody Production and Purification

Goats (La Mancha or Toggenburg breed) were immunized with purifiedrecombinant human full-length proBNP (rhproBNP). A primary intramuscularinjection at multiple sites of 500 ug purified rhproBNP emulsified inComplete Freund's Adjuvant was administered, followed by bi-weekly 250ug intramuscular injections at multiple sites of the purified rhproBNPemulsified in Freund's incomplete adjuvant. The titer of immunized goatswas monitored routinely by screening serum using a half-sandwich ELISAtechnique.

Polyclonal antibodies (PAb) specific for amino acid sequences withinproBNP (1-25, 26-51, 52-76 or 77-108) of Sequence ID No. 1 weresubsequently purified from goat serum by sequential affinitypurification using cyanogen bromide activated sepharose-4B (Pharmacia)coupled, according to the supplier's recommendations, to the followingproteins or peptide sequences:

human IgG (Jackson ImmunoResearch) mouse IgG (Jackson ImmunoResearch)proBNP amino acid sequence #1-25 of  Sequence ID No. 1(H P L G S P G S A S D L E T S G L Q E Q  R N H L Q)coupled to Keyhole Limpet Haemocyanin (ADI Inc.) ORproBNP amino acid sequence #26-51 of Sequence ID No. 1(G K L S E L Q V E Q T S L E P L Q E S P R P T G V W)coupled to Keyhole Limpet Haemocyanin (ADI Inc.) ORproBNP amino acid sequence #52-76 of Sequence ID No. 1(K S R E V A T E G I R G H R K M V L Y T L R A P R)coupled to Keyhole Limpet Haemocyanin (ADI Inc.) ORproBNP amino acid sequence #77-108 of Sequence ID No. 1(BNP-32, S P K M V Q G S G C F G R K M DR I S S S S G L G C K V L R R H) coupled to Keyhole Limpet Haemocyanin(ADI Inc.)

The purified polyclonal antibodies were dialyzed against 20 mM PBS, pH7.4, concentrated by ultrafiltration and stored at −20° C.

Expression of Recombinant Human NT-proBNP

In order to obtain material for use in calibration of a quantitativemethod for measurement of human NT-proBNP, recombinant human NT-proBNP(or rhNT-proBNP) was expressed and purified. A proBNP-pUC9 plasmidconstruct was obtained from Dr. Adolfo J. de Bold (Ottawa HeartInstitute). The rhNT-proBNP ORF was obtained by PCR and subcloning intopET32c (NcoI/XhoI). The sequence at the N-terminus of the rhNT-proBNPORF consisted of thioredoxin, poly-histidine, and S-tag tags, as well asthrombin and enterokinase cleavage sites. There was no extra sequence atthe C-terminus. The protein was expressed in Escherichia coli BL21 (DE3)cells and the crude cellular extract was prepared in non-denaturingconditions. The subsequent affinity purification was completed by Ni-NTAchromatography following the supplier's recommendations.

Screening of Monoclonal Antibodies

Monoclonal antibodies, secreted by hybridoma cell lines hereindesignated as 6G11-F11-D12 and as 1C3-E11-H9 for use in a method ofimmunoassay, wherein said antibodies are specific to the polypeptideconsisting of amino acids 1-25 of human N-terminal brain natriureticfactor BNP(1-25), were obtained from Dr. Adolfo J. De Bold. Thesemonoclonals were produced from supernatants for use in an NT-proBNPELISA in pairing with the instantly described Goat PolyclonalAntibodies, and are designated 6G11 and 1C3 respectively. These clonesare the subject of U.S. Ser. No. 10/299,606 filed on even date herewith,the contents of which are herein incorporated by reference, and weredeposited, in accordance with the Budapest Treaty, with the AmericanType Culture Collection, 10801 University Blvd., Manassas, Va.20110-2209 on Dec. 5, 2002 under Accession Numbers PTA-4844 and PTA-4845respectively. In accordance with 37 CFR 1.808, the depositors assurethat all restrictions imposed on the availability to the public of thedeposited materials will be irrevocably removed upon the granting of apatent. The depositors additionally assure that the deposited materialswill be replaced if viable samples cannot be dispensed by thedepository.

Screening was conducted for:

i) Potential Capture MAb(s) with Goat PAb as Detector

Confluent hybridoma culture supernatants were added to 96-wellmicrotiter plates (NUNC, MaxiSorp, GIBCO BRL) coated with donkeyanti-mouse IgG_((H+L)) immunoglobulins (Jackson ImmunoResearch) at 2μg/ml in 100 mM carbonate buffer, pH 9.6. Excess binding sites wereblocked with bovine serum albumin (BSA) in PBS, pH 7.4. After washingthe plate with wash buffer (PBS containing 0.05% (v/v) Tween 20), 50 μLof each culture supernatant containing monoclonal antibody was incubatedon the plate. Following 1 hour incubation at 37° C. in a CO₂ incubator,the plate was washed with wash buffer. Recombinant human proBNP (Syn-XPharma) was then added to the plate at concentrations of 3 ng/ml or 0ng/ml, and the plate incubated for 2 hours at room temperature (RT) on ashaker. After washing the plate, biotinylated goat polyclonal antibodiesaffinity purified against proBNP amino acid peptide sequences 1-25,26-51 or 52-76 (Syn-X Pharma), diluted appropriately in PBS with 0.5%(w/v) BSA, were added to the appropriate wells. Goat polyclonalantibodies were biotinylated using a Biotin Labeling Kit from Rochefollowing the manufacturer's recommendations. After 1 hour incubation atRT on a shaker, the plate was washed and HRP-conjugated streptavidin(Jackson ImmunoResearch) at a dilution of 1/5000 was added and incubatedfor 1 hour at RT on a shaker. Following washing, TMB substrate solution(Moss) was added and after 8 minutes incubation at RT in the dark, thereaction was stopped with 1 N H₂SO₄ and optical density read at450_(nm). Clones were selected for ascites production based on abilityto pair with the respective goat polyclonal antibody to produce aspecific high intensity signal in wells containing proBNP antigen, andminimal signal in wells containing no proBNP antigen.

ii) Potential Detector MAb(s) with Goat PAb as Capture

96-well microtiter plates were coated with goat polyclonal antibodiesaffinity purified against proBNP amino acid peptide sequences 1-25,26-51, or 52-76 (Syn-X Pharma) at 1 μg/ml in 100 mM carbonate buffer, pH9.6. Excess binding sites were blocked as for method (i). After washingwith wash buffer, recombinant human proBNP (Syn-X Pharma) was added tothe wells at concentrations of 3 ng/ml or 0 ng/ml and the plateincubated for 2 hours at RT on a shaker. Following washing, confluenthybridoma culture supernatants containing monoclonal antibodies wereadded (50 μL per well) and the plates incubated for 1 hour at 37° C. ina CO₂ incubator. After another wash step, HRP conjugated donkeyanti-mouse IgG_((H+L)) (Jackson ImmunoResearch) at a dilution of 1/5000was added to the plate and incubated for 1 hour at RT on a shaker. TMBsubstrate was added, after washing, and the plates developed as formethod (i). Clones were selected for ascites production based on abilityto pair with the respective goat polyclonal antibody to produce aspecific high intensity signal in wells containing proBNP antigen, andminimal signal in wells without antigen.

Final Selection of 6G11 Monoclonal Antibody

Following production of the selected monoclonal antibodies by ascites,and subsequent purification by Protein G (Pharmacia) using knownprocedures, the purified antibodies were retested as described above forscreening of hybridoma supernatants, but for the fact that the purifiedmonoclonal antibodies were appropriately diluted in 100 mM carbonatebuffer, pH 9.6 and coated directly onto the plate for screening ascaptures, or appropriately diluted in PBS containing 0.5% (w/v) BSA forscreening as detectors.

Optimal ELISA specificity and sensitivity for recombinant human proBNPand recombinant human NT-proBNP were obtained using the combination ofgoat polyclonal antibody affinity purified against proBNP amino acidpeptide sequence 26-51 as capture with MAb clone designate 6G11 asdetector. Now referring to FIG. 5, the procedure for carrying out theELISA assay of the instant invention is set forth.

Subsequent analysis of the data derived from human plasma samples testedin accordance with these procedures have demonstrated the utility ofthis antibody combination for yielding excellent sensitivity andspecificity when measuring NT-proBNP levels in apparently healthyindividuals versus heart failure patients.

In accordance with this invention, an ELISA Test Kit is provided for thepurpose of carrying out the above-outlined procedure.

Reagents Supplied

Anti-NT-proBNP Protein Coated Microtitration Strips

One stripholder containing 96 microtitration wells coated with goatpolyclonal anti-NT-proBNP antibody. Store at 2-8° C., in the pouch withdesiccant, until expiry.

NT-proBNP Protein Standards

Six vials, each containing one of the following standards: 0, 50, 150,375, 1500, and 3000 pg/ml of NT-proBNP, are provided. Each vial contains0.5 ml, except for the 0 pg/ml standard which contains 1.0 ml. The extravolume allows for diluting samples that have values greater than 3000pg/ml, if retesting is desired. Store at −70±10° C. Kept at thistemperature, the standards are stable for at least 3 cycles offreeze/thaw and up to 6 months.

NT-proBNP Protein Controls

Two vials, 0.5 ml each, containing NT-proBNP controls at low and highprotein concentration. Store at −70±10° C. Kept at this temperature, thecontrols are stable for at least 3 cycles of freeze/thaw and up to 6months.

Incubation Buffer

One vial containing 10 ml of incubation buffer. Store at 2-8° C. untilexpiry.

Detector Antibody

One vial containing 10 ml of monoclonal anti-NT-proBNP antibody. Storeat 2-8° C. until expiry.

Horseradish Peroxidase (HRP) Conjugate

One vial containing 10 ml of donkey anti-mouse immunoglobulins labeledwith horseradish peroxidase. Store at 2-8° C. until expiry.

Chromogen Solution

One vial containing 10 ml of 3, 3′, 5, 5′-tetramethylbenzidine (TMB)substrate solution. Store at 2-8° C. until expiry.

Wash Concentrate

One bottle containing 60 ml phosphate buffered saline with nonionicdetergent. Dilute contents 25 fold with deionized water before use.Store at 2-8° C.

Stopping Solution

One bottle containing 10 ml 1N sulfuric acid. Store at 2-8° C.

Assay Procedure

In carrying out the assay, the time between addition of samples,standards, and controls to the first well and the last well should notexceed 10 minutes. For large series of samples, run the ELISA in smallbatches to accommodate this time frame.

Mark the microplate wells to be used.

Add 50 μl of the incubation buffer to each well using a semi-automaticpipette.

Using a precision micropipette, add 50 μl of each test sample, NT-proBNPstandard, or NT-proBNP control to the appropriate microwell. In order toensure standard curve consistency, the following order of addition tothe plate is recommended:

Test samples

NT-proBNP standards

NT-proBNP controls

It is recommended that NT-proBNP standards and controls be assayed induplicate.

Cover microwells using an adhesive plate cover and incubate for 2 hourson an orbital microplate shaker at room temperature.

Aspirate and wash each microwell three times with the wash solutionusing an appropriate microplate washer. Blot dry by inverting the plateon absorbent material.

Since incomplete washing adversely affects assay precision, the use ofan automatic microplate washer is highly recommended. Alternatively, ifan automatic microplate washer is not available, washing can beaccomplished manually by repeatedly aspirating microwell contents andrefilling each microwell with 340 μl of wash solution, three times.

Add 100 μl of detector antibody solution to each well using asemi-automatic pipette.

Incubate the wells for 1 hour on an orbital microplate shaker at roomtemperature.

Aspirate and wash microwells three times with the wash solution using anappropriate microplate washer. Blot dry by inverting the plate onabsorbent material.

Add 100 μl of HRP conjugate solution to each well using a semi-automaticpipette.

Cover microwells using an adhesive plate cover and incubate for 30minutes on an orbital microplate shaker at room temperature.

Aspirate and wash microwells three times with wash solution. Blot dry byinverting the plate on absorbent material.

Add 100 μl of the TMB solution to each well using a semi-automaticpipette.

Incubate the wells in the dark for 5 minutes at room temperature. Avoidexposure to direct sunlight.

Add 100 μl of stopping solution (1N sulfuric acid) to each well using asemi-automatic pipette.

Measure the absorbance of the solution in the microwells using amicroplate reader at 450 nm.

Calculation of Results

Calculate the mean absorbance for each well containing standard, controlor test subject plasma.

Plot the mean absorbance reading for each of the standards along they-axis (quadratic) versus the NT-proBNP concentration, in pg/ml, alongthe x-axis (linear).

Draw the best fitting standard curve through the mean of the duplicatepoints.

Determine the NT-proBNP concentrations of the test subjects' plasma andcontrols by interpolating from the standard curve.

Subject plasma specimens reading lower than the lowest standard shouldbe reported as such.

Alternatively, a computer program may be used for handling ELISA typedata to evaluate the NT-proBNP concentrations in test subjects' plasmaand controls.

The following data represent an example dose response curve using thisassay:

Standard Dose (pg/ml) Mean OD 450 nm 0 0.046 50 0.095 150 0.178 3750.347 1500 1.161 3000 1.781 Note: These values should not be used inlieu of a standard curve, which should be prepared at the time of assay.Performance Characteristics

In order to insure quality control standards, two controlsdesignated-low and high—provided in the kit must be analyzed in eachassay. It is recommended that each laboratory use additional controlsfor validation of each assay run.

Summary of NT-proBNP clinical data for the Goat Polyclonal-6G11Monoclonal Elisa Assay

Data is available from 209 subjects diagnosed with congestive heartfailure (NYHA Class III and Class IV) and 101 healthy normal controlsubjects. The receiver operating characteristic (ROC) curve is displayedin FIG. 2; an area under the curve (AUC) of 0.974 was obtained, with acorresponding standard error (s.e.) of 0.008. FIG. 3 displays boxplotsof NT-proBNP levels in the control subjects and the heart failuresubjects; at a cutoff level of 165 pg/mL, the diagnostic sensitivitywith respect to the heart failure subjects was 90.4% (with 189 out of209 such subjects with NT-proBNP levels above the cutoff) and thediagnostic specificity with respect to the control subjects was 94.1%(with 95 out of 101 such subjects with NT-proBNP levels below thecutoff).

Comparison with other NT-proBNP and BNP Assays

In the product insert for the Biosite Triage BNP test (Triage© B-TypeNatriuretic Peptide (BNP) Test, Product insert, Biosite Diagnostics,Inc., 2001), a ROC curve analysis on clinical data obtained from 804heart failure subjects and 1286 control subjects revealed an AUC of0.955 (standard error=0.0053). Comparing this AUC with that of theinstantly disclosed NT-proBNP assay, following the procedure of Hanleyand McNeil (Hanley J A and McNeil B J (1982). “The meaning and use ofthe area under a receiver operating characteristic (ROC) curve.”Radiology 143 29-36), one finds that the instantly disclosed NT-proBNPassay has a significantly higher AUC (p<0.001), indicative of superiordiagnostic performance.

Fischer et al. (Fischer Y, Filzmaier K, Stiegler H, Graf J, Fuhs S,Franke A, Janssens U and Gressner A M (2001). “Evaluation of a New,Rapid Bedside Test for Quantitative Determination of B-Type NatriureticPeptide.” Clinical Chemistry 47 591-594.) gave performance datacomparing the Triage BNP test to an NT-proBNP EIA assay from RocheDiagnostics with respect to 93 subjects with underlying cardiac diseaseand suspected heart failure. In distinguishing subjects with decreasedventricular function from those with preserved ventricular function, anAUC of 0.91 (±0.033 s.e.) was obtained for the Triage BNP test, and anAUC of 0.86 (±0.040 s.e.) was obtained for the Roche NT-proBNP assay.Given a reported correlation between the two neurohormone measurementsof r=0.947, and following the method of Hanley and McNeil (Hanley J Aand McNeil B J (1983), “A method of comparing the areas under ReceiverOperating Characteristic curves derived from the same cases.” Radiology148 839-843) for comparing AUC's derived from the same set of cases, onefinds that the Triage BNP test has a significantly higher AUC than thatof the Roche NT-proBNP assay (p=0.005).

Hammerer-Lercher et al. (Hammerer-Lercher A, Neubauer E, Muller S,Pachinger O, Puschendorf B and Mair J (2001). “Head-to-head comparisonof N-terminal pro-brain natriuretic peptide, brain natriuretic peptideand N-terminal pro-atrial natriuretic peptide in diagnosing leftventricular dysfunction.” Clinica Chimica Acta 310 193-197) compared theShionogi IMRA BNP assay to the Biomedica EIA NT-proBNP assay withrespect to the same population of 57 patients with stable chronic heartfailure. In distinguishing subjects with decreased ventricular functionfrom those with preserved ventricular function, an AUC of 0.75 (±0.06s.e.) was obtained for the BNP assay, and an AUC of 0.67 (±0.07 s.e.)was obtained for the Biomedica NT-proBNP assay. Following the method ofHanley and McNeil (Hanley J A and McNeil B J (1983). “A method ofcomparing the areas under Receiver Operating Characteristic curvesderived from the same cases.” Radiology 148 839-843), one finds that theShionogi BNP assay has a significantly higher AUC than that of theBiomedica NT-proBNP assay (p=0.009).

Luchner et al. (Luchner A, Hengstenberg C, Lowel H, Trawinski J, BaumannM, Riegger G, Schunkert H and Holmer S (2002). “N-Terminal Pro-BrainNatriuretic Peptide After Myocardial Infarction.” Hypertension 3999-104) conducted a large clinical study involving 594 myocardialinfarction subjects and 449 healthy controls, in order to determine theability of the Roche EIA NT-proBNP assay to predict decreasedventricular function in these subjects. The authors quoted an AUC of0.77 (±0.057 s.e.) with respect to NT-proBNP in separating subjects witha left ventricular ejection fraction of less than 35% from those with ahigher ejection fraction. This AUC is significantly lower than thatquoted above for the instantly disclosed NT-proBNP assay (p=0.0001).

Thus, on the basis of quantifying the variously available assays fordetermining the presence of NT-proBNp based upon an area under the curveanalysis, the instant assay would be expected to exhibit superiordiagnostic performance.

All patents and publications mentioned in this specification areindicative of the levels of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

It is to be understood that while a certain form of the invention isillustrated, it is not to be limited to the specific form or arrangementherein described and shown. It will be apparent to those skilled in theart that various changes may be made without departing from the scope ofthe invention and the invention is not to be considered limited to whatis shown and described in the specification. One skilled in the art willreadily appreciate that the present invention is well adapted to carryout the objectives and obtain the ends and advantages mentioned, as wellas those inherent therein. The embodiments, methods, procedures andtechniques described herein are presently representative of thepreferred embodiments, are intended to be exemplary and are not intendedas limitations on the scope. Changes therein and other uses will occurto those skilled in the art which are encompassed within the spirit ofthe invention and are defined by the scope of the appended claims.Although the invention has been described in connection with specificpreferred embodiments, it should be understood that the invention asclaimed should not be unduly limited to such specific embodiments.Indeed, various modifications of the described modes for carrying outthe invention which are obvious to those skilled in the art are intendedto be within the scope of the following claims.

What is claimed is:
 1. A kit for quantitative measurement of humanAmino-Terminal Pro-Brain Natriuretic Peptide (NT-proBNP) based on asandwich ELISA technique, comprising: isolated goat polyclonalantibodies obtained by: (1) immunizing goats with purified recombinanthuman full-length proBNP of SEQ ID NO: 1, and (2) affinity purifyingfrom goat serum from the immunized goats against the peptide consistingof amino acids 26-51 of SEQ ID NO: 1; isolated NT-proBNP monoclonalantibodies, wherein said NT-proBNP monoclonal antibodies are specificfor the sequence of amino acid residues that are different from theisolated polyclonal antibodies, wherein the monoclonal antibodies arelabeled with a detector; and a reporter capable of reacting with saiddetector to form a reaction product, wherein the reaction product doesnot diffuse, and wherein the NT-proBNP monoclonal antibodies areproduced by a hybridoma cell line designated as 6G11-F11-D12 anddeposited with the American Type Culture Collection (ATCC) as AccessionNumber PTA 4844 and is specific for a polypeptide consisting of aminoacids 1-25 of SEQ ID NO: 1, wherein the isolated goat polyclonalantibodies are immobilized as a primary binding partner.
 2. The kit ofclaim 1, wherein the detector is biotin.
 3. The kit of claim 1, whereinthe reporter is peroxidase-labeled streptavidin.
 4. The kit of claim 3,wherein the peroxidase is a horseradish peroxidase.
 5. The kit of claim1, wherein the kit is used for predicting morbidity and mortality in acongestive heart failure patient.
 6. The kit of claim 1, wherein thepolyclonal antibodies are attached to a solid support.
 7. A quantitativemethod of measuring human NT-proBNP by the kit of claim 1 for predictingmorbidity and mortality in a congestive heart failure patient,comprising: (a) providing isolated polyclonal antibodies (b) contactinga bodily fluid sample from a patient to said isolated immobilizedpolyclonal antibodies; (c) providing proBNP monoclonal antibodieswherein said proBNP monoclonal antibodies are specific for the sequenceof amino acid residues distinct from the specific sequence for theisolated polyclonal antibodies; (d) effecting an immunoreaction, whereinthe immunoreaction is between a human NT-proBNP in the bodily fluidsample and the monoclonal antibodies wherein the monoclonal antibodiesare labeled with a detector; and (e) detecting said immunoreaction by areporter capable of reacting with said detector to form a reactionproduct, wherein the reaction product does not diffuse, and wherein theNT-proBNP monoclonal antibodies are produced by a hybridoma cell linedesignated as 6G11-F11-D12 and deposited with the American Type CultureCollection (ATCC) as Accession Number PTA 4844 and is specific for apolypeptide consisting of amino acids 1-25 of SEQ ID NO:
 1. 8. Themethod of claim 7, wherein the isolated polyclonal antibodies areselected for being specific to an amino acid sequence consisting ofamino acids 26-51 of SEQ ID NO:
 1. 9. The method of claim 7, wherein themonoclonal antibodies are produced by a hybridoma cell line designatedas 1C3-E11-H9 and deposited with the American Type Culture Collection(ATCC) as Accession Number PTA 4845 4 and is specific for a polypeptideconsisting of amino acids 1-25 of SEQ ID NO:
 1. 10. The method of claim8, wherein the monoclonal antibody further comprises a detector.
 11. Themethod of claim 7, wherein the detecting is direct.
 12. The method ofclaim 7, wherein the detecting is indirect.
 13. The method of claim 7,wherein in step (d) the human NT-proBNP is bound by the polyclonalantibodies.
 14. A kit for quantitative measurement of humanAmino-Terminal Pro-Brain Natriuretic Peptide (NT-proBNP) based on asandwich ELISA technique, comprising: isolated goat polyclonalantibodies obtained by: (1) immunizing goats with purified recombinanthuman full-length proBNP of SEQ ID NO: 1, and (2) affinity purifyingfrom goat serum from the immunized goats against the peptide consistingof amino acids 26-51 of SEQ ID NO: 1; isolated NT-proBNP monoclonalantibodies, wherein said NT-proBNP monoclonal antibodies are specificfor the sequence of amino acid residues that are different from theisolated polyclonal antibodies, wherein the monoclonal antibodies arelabeled with a detector; and a reporter capable of reacting with saiddetector to form a reaction product, wherein the reaction product doesnot diffuse, and wherein the NT-proBNP monoclonal antibodies areproduced by a hybridoma cell line designated as 1C3-E11-H9 and depositedwith the American Type Culture Collection (ATCC) as Accession Number PTA4845 and is specific for a polypeptide consisting of amino acids 1-25 ofSEQ ID NO: 1, wherein the isolated goat polyclonal antibodies areimmobilized as a primary binding partner.
 15. The kit of claim 14,wherein the detector is biotin.
 16. The kit of claim 14, wherein thereporter is peroxidase-labeled streptavidin.
 17. The kit of claim 16,wherein the peroxidase is a horseradish peroxidase.
 18. The kit of claim14, wherein the it is used for predicting morbidity and mortality in acongestive heart failure patient.
 19. The kit of claim 14, wherein thepolyclonal antibodies are attached to a solid support.
 20. Aquantitative method of measuring human NT-proBNP by the kit of claim 14for predicting morbidity and mortality in a congestive heart failurepatient, comprising: (a) providing isolated polyclonal antibodies; (b)contacting a bodily fluid sample from a patient to said isolatedimmobilized polyclonal antibodies; (c) providing proBNP monoclonalantibodies wherein said proBNP monoclonal antibodies are specific forthe sequence of amino acid residues distinct from the specific sequencefor the isolated polyclonal antibodies; (d) effecting an immunoreaction,wherein the immunoreaction is between a human NT-proBNP in the bodilyfluid sample and the monoclonal antibodies wherein the monoclonalantibodies are labeled with a detector; and (e) detecting saidimmunoreaction by a reporter capable of reacting with said detector toform a reaction product, wherein the reaction product does not diffuse,and wherein the NT-proBNP monoclonal antibodies are produced by ahybridoma cell line designated as 1C3-E11-B9 and deposited with theAmerican Type Culture Collection (ATCC) as Accession Number PTA 4845 andis specific for a polypeptide consisting of amino acids 1-25 of SEQ IDNO: 1.